Description
Contents
1.0 BACKGROUND
1.1 WHAT ARE SSTI?
The skin is colonized by microorganisms that have the potential to infiltrate and cause infection when the skin’s barrier function is impaired [1]. Bacteria capable of infiltrating the skin and surrounding tissues can cause a broad spectrum of infectious conditions that affect the skin, subcutaneous tissue, and underlying soft tissue. These diseases are referred to as Skin and Soft Tissue infections (SSTIs) [2]. SSTIs can be predisposed by several factors, including trauma, surgery, underlying skin diseases, diabetes mellitus, cancer, or immunological suppression [3]. SSTIs can range from moderate and self-limiting to severe and progressive, resulting in surrounding tissue necrosis. According to the degree of skin infection, the US Food and Drug Administration (FDA) and the Infectious Diseases Society of America (IDSA) classify SSTIs into two categories: complicated SSTIs (cSSTIs) and uncomplicated SSTIs (also known as superficial infections).[4]. Comparatively, uncomplicated SSTIs include cellulitis, simple abscesses, impetigo, and furuncles, whereas complicated SSTIs comprise deep soft tissue infections such as necrotizing infections, infected ulcers, infected burns, and severe abscesses. [5]. Alternatively, SSTIs can be grouped by the anatomic tissue layers involved (Figure 1). For example, cellulitis affects the dermis and subcutaneous tissue, meanwhile, necrotizing infections commonly affects the deep fascia but can also invade the dermis down through to the muscle [3]. In the United States more than 14million outpatients suffer from SSTIs with 900,000 hospitalized yearly [2]. Data obtained from the Global Burden of Disease (GBD) 2017 revealed that skin diseases contribute to an estimate of 48700-95600 deaths [5].
Figure 1: Classification of SSTIs based on the anatomic tissue layers.
SSTIs are caused by Gram-positive bacteria, including Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pyogenes. Gram-negative bacteria, such as Klebsiella pneumoniae and Escherichia coli, are less frequently linked to SSTIs [4]. Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for more than half of SSTI cases. MRSA is a serious healthcare issue linked to exceptionally high rates of morbidity and death in people who are infected [6].
1.2 Methicillin-resistant Staphylococcus aureus
Staphylococcus aureus is a global threat to public health [7]. It produces an arsenal of virulent factors that enables it to colonize host cells, lyse host tissue, modify host tissue immunity, disseminate throughout the body, form biofilm and produce toxins which are associated with the primary clinical symptoms on the skin [8]. Methicillin is a semisynthetic antibiotic that works by inhibiting infections caused by Staphylococci. Methicillin-resistant Staphylococcus aureus (MRSA) is the name given to S. aureus that shows methicillin resistance. MRSA develops resistance to antibiotics through the acquisition of genes (mobile genetic elements) capable of horizontal gene transfer. For example, mecA or mecC gene encodes for a truncated penicillin binding protein 2A (PBP2A), the enzyme responsible for crosslinking the peptidoglycans of bacterial cell wall. This induces penicillin resistance due to the inability of the protein to bind to penicillin (methicillin) [9].
1.3 Treatment Strategies
The keys to effective treatment include early diagnosis, adequate antibiotic intervention and rapid surgical intervention. [4]. The Surgical Infection Society (SIS) suggests that vancomycin, linezolid (LZD), daptomycin, ceftaroline and telavancin should be used as a first line of treatment of cSSTIs caused by MRSA [5]. Vancomycin is a glycopeptide antibiotic that is typically used to treat cSSTIs caused by MRSA but is also used as a last option for severe infections caused by Gram-positive bacteria [10].
Unlike other antibiotic that mostly inhibit bacteria by binding to protein targets, Vancomycin functions by preventing Gram-positive bacteria from synthesizing their cell walls. In order to accomplish this, it binds to the un-crosslinked Lipid II's C-terminal d-Ala-d-Ala moiety, an intermediary in the formation of the peptidoglycan layer [10]. This binding obstructs the ability of penicillin-binding proteins (PBPs) to cross-link Lipid II into mature peptidoglycan and thus compromises the integrity of bacterial cell membrane, leading to osmotic stress and bursting of the cell [10]. The use of vancomycin as a therapy has a number of limitations. Vancomycin can only be administered intravenously, which is a very invasive method, it has a low tissue penetration and nephrotoxicity risk throughout treatment. Resent years have also seen the emergence of Staphylococcus aureus isolates that are completely resistant to vancomycin. This resistance is mediated by vanA gene clusters, a transposable element transferred from vancomycin-resistant enterococcus.
2.0 Antimicrobial peptides (AMPs)
Due to high rates of treatment failure, relapse, and the ability of microorganisms to develop resistance, together with the indiscriminate use of antibiotics, treatment of SSTIs is becoming more difficult. There is an urgent need for novel antimicrobial candidates due to the increasing frequency of antibiotic resistance worldwide. Natural antimicrobial peptides (AMPs) derived from different species of organisms ranging from animal to plants are a very desirable resource in the hunt of novel antimicrobial medications as they have biological effects against a wide range of drug-resistant pathogenic bacteria, can act as important regulators of the innate immune system and tend to reduce resistance development. Antimicrobial Peptides (AMPs) are mostly positively charged, hydrophobic or amphipathic low molecular weight oligopeptides containing 5 to 100 amino acid residues in a linear or cyclic arrangement [11], [12]. AMPs are ribosomally synthesized by all species of life as a first line of defense against invasion by microorganisms [13]. AMPs have a broad-spectrum antibacterial, antibiofilm, anticancer and immunomodulatory effects on both Gram-positive and Gram-negative bacteria, as well as parasites, viruses, fungi, protozoa, viruses and even tumors [14] [11], [15]. AMPs are soluble in aqueous environments due to their hydrophobic and hydrophilic side chain [16]. Since the discovery of the first AMP gramicidin from Bacillus sp. By Dubus in 1939 and its ability to protect mice from pneumococcal infection [11] several other AMPs have been discovered in both prokaryotes and eukaryotes [17]. Until 2018, over 4800 peptides (4256 natural peptides and 593 synthetic peptides) have been reported in the AMP repository. AMPs have unique features that that contributes to their value as therapeutic agents, one of which includes their ability to rapidly kill on initial contact with the cell membrane. Since amino acids make up AMPs, it is simple to chemically manufacture AMPs, produce them using recombinant expression systems, or alter the structure of already-existing AMPs to increase their stability[16].
2.1 Structure and Mode of action of AMPs
AMPs share a net positive charge at neutral pH, and an amphipathic character in hydrophobic environment[18]. AMPs are grouped into four main subgroups based on their secondary structures which includes β-sheet, α-helix (which are the most encountered), extended, and loop[13], [19]. The α-helix are the most studied amongst the AMPs [11]. The size, sequence, charge, structure and conformation, hydrophobicity, and amphipathic qualities of AMPs are among the several variables that influence their mechanism. Because of their intricate mode of action, AMPs can affect several intracellular targets as well as the cell wall and membrane [20].
AMPs exerts its antibacterial effects on cell wall mainly by interfering with cell wall synthesis or by destroying the cell wall structure. The thick cell walls of gram-positive bacteria contains 15–50 layers of peptidoglycan (PGN), which is essential to the survival and integrity of the bacterium. By attaching to lipid II, a precursor for the formation of the cell wall and a crucial element in the manufacture of PGN, AMPs can impede the biosynthesis of cell wall. By preventing the formation of teichoic acid lipid III, a precursor to cell wall teichoic acid, AMPs can also compromise the integrity of the cell wall. Gram-negative bacteria can be inhibited by AMPs acting on their outer membrane in a number of ways, such as by aggregating lipopolysaccharide (LPS) micelles and neutralizing the charge on the outer membrane.[20].
Like the cell wall, bacterial cell membrane is also an important target of most AMPs. AMPs binds to bacterial cell membrane through physicochemical action, continuous accumulation on the surface of the cell membrane. Upon reaching a certain concentration threshold, AMPs increases the permeability of the cell membrane leading to membrane lysis and release of the cytoplasmic contents, thereby exerting antibacterial activity. Interestingly, AMPs have no effect on mammalian cell membrane due to the difference in the cell membrane composition. Three models have been used to describe how AMPs interact with cell membranes, they include the barrel-stave model, toroidal-pore model, and the carpet model. In the barrel-stave model, helical polypeptides accumulate within the cell wall and are injected vertically into the lipid bilayer to create bundle-like pores in the membrane, which in turn alters the membrane's permeability. Peptides interact with the lipid head groups in the toroidal-pore model, causing the bilayer to bend and insert vertically into the membrane bilayer, forming annular holes made up of phospholipid head bundles and peptides. In the carpet model, AMPs interact with anionic phospholipid head groups present on the membrane surface electrostatically. The phospholipid bilayer gets destroyed by micelle production at high peptide concentrations.
AMPs can also enter the cell membrane by endocytosis, directly translocate through membrane boundary defects, or through receptor-mediated transport pathways to exert their anti-bacterial effects on intracellular targets. 1) They have the ability to disrupt nucleic acid repair pathways and break down nucleic acid conformation, which prevents the creation of proteins, RNA, or DNA. 2) They inhibit the activity of enzymes/proteins in nucleic acid and protein synthesis metabolic pathways. 3) AMPs can also exert an effect against the ribosome to inhibit protein synthesis. 4) Inhibit cell division and blockage of the cell cycle. And 5) Interfere with the proper folding and assembly of proteins.
Figure 2: Mechanism of AMPs[21]
2.2 Antimicrobial peptides for the treatment of skin and soft tissue infection
Numerous natural and artificially produced antimicrobial peptides (AMPs) have been studied for their potential use in the treatment of SSTIs. Histatin, a naturally occurring human salivary peptide, peptide LL-37 produced by epithelial and immune cells, human ApoB derived peptides, the synthetic peptide PXL150, GK-19, a novel AMP derived from Scorpion venom-derived peptide Androctonus amoreuxi AMP1 (AamAP1), and AMC-109, a synthetic cationic AMP, are among them [22][23][24][25][26]. These peptides not only suppress pathogens but also affect cell proliferation and differentiation, increase angiogenesis, interact with lipopolysaccharides, and modify the expression of pro and anti-inflammatory cytokines [24]. Broad range microbicidal efficacy against both Gram-positive and Gram-negative bacteria, including resistant strains, has been established in vitro by the synthetic AMPs PXL150 and AMC-109[22], [25]. In addition to inhibiting both Gram-positive and Gram-negative bacteria, the AMP GK-19 also killed fungus by rupturing their microbial cell membrane. When administered topically to a model of SSTI-infected mice, it also showed signs of healing [26]. Overall, this study demonstrates that AMPs could be promising drug candidates for the treatment of SSTIs caused by drug resistant bacteria including MRSA.
3.0 Our solution
Despite the advantages conferred by AMPs, there are still some challenges that need to be overcome such as susceptibility of AMPs to harsh environmental conditions including extreme temperature, pH and degradation by proteolytic enzymes, hydrolysis, oxidation photolysis, minimal residence time as well as high costs. Bacteria inhibiting extreme ecological niches such as hot springs exhibit unique physical and structural characteristics that enable them to survive in intense environmental conditions and give them the potential to produce stable secondary metabolites including AMPs. Thermophilic bacteria inhibiting hot springs are not fully explored in terms of AMPs. However, few reports have demonstrated that AMPs produced by thermophilic bacteria has displayed potent activity against strains of Gram-positive bacteria including Staphylococcus aureus. Thus, AMPs from thermophilic bacteria may act as templates for the design of more potent and stable AMPs that are effective against multidrug resistant bacteria including the MRSA responsible for SSTIs. The goal of our research is to produce novel AMPs expressed by thermophilic bacteria isolated from hot springs in Saudi Arabia, believed to possess some therapeutic advantages. Genes encoding the AMPs will be synthesized, heterologously expressed in Escherichia coli, purified, and tested against staphylococcus aureus. To enhance the stability, prolong delivery, and optimize the effectiveness of our purified AMPs at the site of an infection, the AMP will be formulated into topical creams or ointment that can be used to manage SSTIs.
4.0 Project Inspiration
The motivation of this project stems from the prevalence in antimicrobial resistance and the urgent need for novel therapeutics that can tackle this rising issue. We choose skin and soft tissue infection as it is the most common medical condition encountered in clinical practice and because Staphylococcus aureus particularly the methicillin-resistant Staphylococcus aureus is the primary pathogen associated with SSTIs. MRSA poses a significant challenge in the treatment of SSTIs due to its ability to resist multiple antibiotics. Researching alternative treatment, such as antimicrobial peptides can help combat antibiotic resistance and improve treatment options. Also, the delivery method of available therapeutics is highly invasive and is associated with several side effects. This has inspired our team to produce AMPs encoded by thermophilic bacteria which are known to produce peptides that are stable, has reduced susceptibility to proteolytic degradation and has activity against multidrug-resistant pathogen. We have also decided to develop our AMPs into a topical formulation that can be applied directly to the infected site to reduce the risk of adverse side effects posed by other treatment methods.
References
M. S. Linz, A. Mattappallil, D. Finkel, and D. Parker, “Clinical Impact of Staphylococcus aureus Skin and Soft Tissue Infections,” Antibiotics, vol. 12, no. 3. MDPI, Mar. 01, 2023. doi: 10.3390/antibiotics12030557.
W. Liang, H. Yin, H. Chen, J. Xu, and Y. Cai, “Efficacy and safety of omadacycline for treating complicated skin and soft tissue infections: a meta-analysis of randomized controlled trials,” BMC Infect Dis, vol. 24, no. 1, Dec. 2024, doi: 10.1186/s12879-024-09097-3.
B. Silverberg, “A structured approach to skin and soft tissue infections (SSTIs) in an ambulatory setting,” Clinics and Practice, vol. 11, no. 1. Page Press Publications, pp. 65–74, Mar. 01, 2021. doi: 10.3390/clinpract11010011.
A. F. Cardona and S. E. Wilson, “Skin and Soft-Tissue Infections: A Critical Review and the Role of Telavancin in Their Treatment,” Clinical Infectious Diseases, vol. 61, pp. S69–S78, Sep. 2015, doi: 10.1093/cid/civ528.
N. Mohamed, R. R. Valdez, C. Fandiño, M. Baudrit, D. R. Falci, and J. D. C. Murillo, “In vitro activity of ceftaroline against bacterial isolates causing skin and soft tissue and respiratory tract infections collected in Latin American countries, ATLAS program 2016–2020,” J Glob Antimicrob Resist, vol. 36, pp. 4–12, Mar. 2024, doi: 10.1016/j.jgar.2023.11.006.
S. A. Alhunaif et al., “Methicillin-Resistant Staphylococcus Aureus Bacteremia: Epidemiology, Clinical Characteristics, Risk Factors, and Outcomes in a Tertiary Care Center in Riyadh, Saudi Arabia,” Cureus, May 2021, doi: 10.7759/cureus.14934.
C. J. Murray et al., “Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis,” The Lancet, vol. 399, no. 10325, pp. 629–655, Feb. 2022, doi: 10.1016/S0140-6736(21)02724-0.
P. Del Giudice, “Skin infections caused by staphylococcus aureus,” Acta Derm Venereol, vol. 100, no. 100-year theme Cutaneous and genital infections, pp. 208–215, 2020, doi: 10.2340/00015555-3466.
Y. Cong, S. Yang, and X. Rao, “Vancomycin resistant Staphylococcus aureus infections: A review of case updating and clinical features,” Journal of Advanced Research, vol. 21. Elsevier B.V., pp. 169–176, Jan. 01, 2020. doi: 10.1016/j.jare.2019.10.005.
P. J. Stogios and A. Savchenko, “Molecular mechanisms of vancomycin resistance,” Protein Science, vol. 29, no. 3. Blackwell Publishing Ltd, pp. 654–669, Mar. 01, 2020. doi: 10.1002/pro.3819.
A. A. Bahar and D. Ren, “Antimicrobial peptides,” Pharmaceuticals, vol. 6, no. 12, pp. 1543–1575, 2013, doi: 10.3390/ph6121543.
S. Chakraborty, R. Chatterjee, and D. Chakravortty, “Evolving and assembling to pierce through: Evolutionary and structural aspects of antimicrobial peptides,” Comput Struct Biotechnol J, vol. 20, pp. 2247–2258, 2022, doi: 10.1016/j.csbj.2022.05.002.
P. Kumar, J. N. Kizhakkedathu, and S. K. Straus, “Antimicrobial peptides: Diversity, mechanism of action and strategies to improve the activity and biocompatibility in vivo,” Biomolecules, vol. 8, no. 1, 2018, doi: 10.3390/biom8010004.
R. K. Thapa, D. B. Diep, and H. H. Tønnesen, “Topical antimicrobial peptide formulations for wound healing: Current developments and future prospects,” Acta Biomaterialia, vol. 103. Acta Materialia Inc, pp. 52–67, Feb. 01, 2020. doi: 10.1016/j.actbio.2019.12.025.
E. Rachmawati, S. Asarina, G. B. Kennardi, R. Safitri, T. Subroto, and A. M. Maskoen, “Antimicrobial peptide coding gene of thermophilic bacteria isolated from crater hot spring in mountains around West Java,” J Appl Biol Biotechnol, vol. 11, no. 2, pp. 220–225, 2023, doi: 10.7324/JABB.2023.110224.
J. K. Boparai and P. K. Sharma, “Mini Review on Antimicrobial Peptides, Sources, Mechanism and Recent Applications,” Protein Pept Lett, vol. 27, no. 1, pp. 4–16, 2019, doi: 10.2174/0929866526666190822165812.
B. S. da Silva, A. Díaz-Roa, E. S. Yamane, M. A. F. Hayashi, and P. I. Silva Junior, “Doderlin: isolation and characterization of a broad-spectrum antimicrobial peptide from Lactobacillus acidophilus,” Res Microbiol, vol. 174, no. 3, p. 103995, 2023, doi: 10.1016/j.resmic.2022.103995.
A. Di Grazia et al., “D-Amino acids incorporation in the frog skin-derived peptide esculentin-1a(1-21)NH2 is beneficial for its multiple functions,” Amino Acids, vol. 47, no. 12, pp. 2505–2519, Dec. 2015, doi: 10.1007/s00726-015-2041-y.
L. J. Zhang and R. L. Gallo, “Antimicrobial peptides,” Current Biology, vol. 26, no. 1, pp. R14–R19, 2016, doi: 10.1016/j.cub.2015.11.017.
X. Li, S. Zuo, B. Wang, K. Zhang, and Y. Wang, “Antimicrobial Mechanisms and Clinical Application Prospects of Antimicrobial Peptides,” Molecules, vol. 27, no. 9, May 2022, doi: 10.3390/molecules27092675.
A. Falanga et al., “Marine antimicrobial peptides: Nature provides templates for the design of novel compounds against pathogenic bacteria,” International Journal of Molecular Sciences, vol. 17, no. 5. MDPI AG, May 21, 2016. doi: 10.3390/ijms17050785.
J. Håkansson, J. P. Cavanagh, W. Stensen, B. Mortensen, J. S. Svendsen, and J. Svenson, “In vitro and in vivo antibacterial properties of peptide AMC-109 impregnated wound dressings and gels,” Journal of Antibiotics, vol. 74, no. 5, pp. 337–345, May 2021, doi: 10.1038/s41429-021-00406-5.
A. Gomes, C. Teixeira, R. Ferraz, C. Prudencio, and P. Gomes, “Wound-healing peptides for treatment of chronic diabetic foot ulcers and other infected skin injuries,” Molecules, vol. 22, no. 10. MDPI AG, Oct. 01, 2017. doi: 10.3390/molecules22101743.
A. Ramata-Stunda et al., “Comparative Evaluation of Existing and Rationally Designed Novel Antimicrobial Peptides for Treatment of Skin and Soft Tissue Infections,” Antibiotics, vol. 12, no. 3, Mar. 2023, doi: 10.3390/antibiotics12030551.
E. Myhrman, J. Håkansson, K. Lindgren, C. Björn, V. Sjöstrand, and M. Mahlapuu, “The novel antimicrobial peptide PXL150 in the local treatment of skin and soft tissue infections,” Appl Microbiol Biotechnol, vol. 97, no. 7, pp. 3085–3096, Apr. 2013, doi: 10.1007/s00253-012-4439-8.
C. Song et al., “Antibacterial and Antifungal Properties of a Novel Antimicrobial Peptide GK-19 and Its Application in Skin and Soft Tissue Infections Induced by MRSA or Candida albicans,” Pharmaceutics, vol. 14, no. 9, Sep. 2022, doi: 10.3390/pharmaceutics14091937.